NK exosome isolation by Size Exclusion Chromatography

For NK exosome preparation, NK cells were cultured for 48h in 10% FBS/RPMI medium that had been cleared of bovine exosomes by an ultracentrifugation step for 1h at 100,000g. The supernatant was centrifuged at 300g for 10 minutes, followed by centrifugation at 3000g for 10 minutes. The clarified supernatant was then concentrated to approximately 500μl on a 100KD Amicon Ultra Centrifugal filter (Millipore). The NK exosomes were then isolated from the concentrated supernatant by size exclusion chromatography (SEC). Sephacryl S-300 High Resolution (GE Healthcare) was packed on a glass econo-column chromatography column (BioRad) (10cm height, 1.5cm diameter). The column was washed with 0.32% Sodium Citrate in PBS and the supernatant was loaded onto the column and allowed to enter the resin by gravity flow. The eluate was collected in 23 fractions of 15 drops (~500μl) on a Model 2110 Fraction Collector (BioRad). For each fraction, the presence of exosomes was determined by nanoparticle tracking analysis, protein concentration and anti-CD81, anti-Calnexin, anti-TSG101, and anti-Fibronectin immunoblotting. The exosome-containing fractions were then further concentrated to 1/100^th of the original supernatant.